Detection of SARS-CoV-2 in Terrestrial Animals in Southern Nigeria: Potential Cases of Reverse Zoonosis.

Since SARS-CoV-2 caused the COVID-19 pandemic, records have suggested the occurrence of reverse zoonosis of pets and farm animals in contact with SARS-CoV-2-positive humans in the Occident. However, there is little information on the spread of the virus among animals in contact with humans in Africa. Therefore, this study aimed to investigate the occurrence of SARS-CoV-2 in various animals in Nigeria. Overall, 791 animals from Ebonyi, Ogun, Ondo, and Oyo States, Nigeria were screened for SARS-CoV-2 using RT-qPCR (n = 364) and IgG ELISA (n = 654). SARS-CoV-2 positivity rates were 45.9% (RT-qPCR) and 1.4% (ELISA). SARS-CoV-2 RNA was detected in almost all animal taxa and sampling locations except Oyo State. SARS-CoV-2 IgGs were detected only in goats from Ebonyi and pigs from Ogun States. Overall, SARS-CoV-2 infectivity rates were higher in 2021 than in 2022. Our study highlights the ability of the virus to infect various animals. It presents the first report of natural SARS-CoV-2 infection in poultry, pigs, domestic ruminants, and lizards. The close human-animal interactions in these settings suggest ongoing reverse zoonosis, highlighting the role of behavioral factors of transmission and the potential for SARS-CoV-2 to spread among animals. These underscore the importance of continuous monitoring to detect and intervene in any eventual upsurge.

Structural changes in the SARS-CoV-2 spike E406W mutant escaping a clinical monoclonal antibody cocktail.

Continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is eroding antibody responses elicited by prior vaccination and infection. The SARS-CoV-2 receptor-binding domain (RBD) E406W mutation abrogates neutralization mediated by the REGEN-COV therapeutic monoclonal antibody (mAb) COVID-19 cocktail and the AZD1061 (COV2-2130) mAb. Here, we show that this mutation remodels the receptor-binding site allosterically, thereby altering the epitopes recognized by these three mAbs and vaccine-elicited neutralizing antibodies while remaining functional. Our results demonstrate the spectacular structural and functional plasticity of the SARS-CoV-2 RBD, which is continuously evolving in emerging SARS-CoV-2 variants, including currently circulating strains that are accumulating mutations in the antigenic sites remodeled by the E406W substitution.

Dynamics of SARS-CoV-2 VOC Neutralization and Novel mAb Reveal Protection against Omicron.

New variants of SARS-CoV-2 continue to emerge and evade immunity. We isolated SARS-CoV-2 temporally across the pandemic starting with the first emergence of the virus in the western hemisphere and evaluated the immune escape among variants. A clinic-to-lab viral isolation and characterization pipeline was established to rapidly isolate, sequence, and characterize SARS-CoV-2 variants. A virus neutralization assay was applied to quantitate humoral immunity from infection and/or vaccination. A panel of novel monoclonal antibodies was evaluated for antiviral efficacy. We directly compared all variants, showing that convalescence greater than 5 months post-symptom onset from ancestral virus provides little protection against SARS-CoV-2 variants. Vaccination enhances immunity against viral variants, except for Omicron BA.1, while a three-dose vaccine regimen provides over 50-fold enhanced protection against Omicron BA.1 compared to a two-dose. A novel Mab neutralizes Omicron BA.1 and BA.2 variants better than the clinically approved Mabs, although neither can neutralize Omicron BA.4 or BA.5. Thus, the need remains for continued vaccination-booster efforts, with innovation for vaccine and Mab improvement for broadly neutralizing activity. The usefulness of specific Mab applications links with the window of clinical opportunity when a cognate viral variant is present in the infected population.

A comparative study of eight serological methods shows that spike protein-based ELISAs are the most accurate tests for serodiagnosing SARS-CoV-2 infections in cats and dogs.

Introduction: Coronavirus disease 2019 (COVID-19) is an infectious zoonotic disease caused by SARS-CoV-2. Monitoring the infection in pets is recommended for human disease surveillance, prevention, and control since the virus can spread from people to animals during close contact. Several diagnostic tests have been adapted from humans to animals, but limited data on the validation process are available. Methods: Herein, the first comparative study of six "in house" and two commercial serological tests developed to monitor SARS-CoV-2 infection in pets was performed with a well-coded panel of sera (61 cat sera and 74 dog sera) with a conservative criterion (viral seroneutralisation and/or RT-qPCR results) as a reference. Four "in house" tests based on either the RBD fragment of the spike protein (RBD-S) or the N-terminal fragment of the nucleoprotein (N) were developed for the first time. The analytical specificity (ASp) of those tests that showed the best diagnostic performance was assessed. The validation included the analysis of a panel of sera obtained pre-pandemic from cats and dogs infected with other coronaviruses to determine the analytical Sp (17 cat sera and 41 dog sera). Results and discussion: ELISAS based on the S protein are recommended in serosurveillance studies for cats (RBD-S SALUVET ELISA, ELISA COVID UNIZAR and INgezim® COVID 19 S VET) and dogs (INgezim® COVID 19 S VET and RBD-S SALUVET ELISA). These tests showed higher diagnostic sensitivity (Se) and DSp in cats (>90%) than in dogs. When sera obtained prior to the pandemic and from animals infected with other coronaviruses were analyzed by RBD-S and N SALUVET ELISAs and INgezim® COVID 19 S VET, a few cross reactors or no cross reactions were detected when dog and cat sera were analyzed by tests based on the S protein, respectively. In contrast, the number of cross reactions increased when the test was based on the N protein. Thus, the use of tests based on the N protein was discarded for serodiagnosis purposes. The results obtained revealed the most accurate serological tests for each species. Further studies should attempt to improve the diagnostic performance of serological tests developed for dogs.

SARS-CoV-2 spike conformation determines plasma neutralizing activity elicited by a wide panel of human vaccines.

Numerous safe and effective COVID-19 vaccines have been developed worldwide that utilize various delivery technologies and engineering strategies. We show here that vaccines containing prefusion-stabilizing S mutations elicit antibody responses in humans with enhanced recognition of S and the S1 subunit relative to postfusion S, as compared to vaccines lacking these mutations or natural infection. Prefusion S and S1 antibody binding titers positively and equivalently correlated with neutralizing activity and depletion of S1-directed antibodies completely abrogated plasma neutralizing activity. We show that neutralizing activity is almost entirely directed to the S1 subunit and that variant cross-neutralization is mediated solely by RBD-specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants than current technologies.

Emergence and phenotypic characterization of the global SARS-CoV-2 C.1.2 lineage.

Global genomic surveillance of SARS-CoV-2 has identified variants associated with increased transmissibility, neutralization resistance and disease severity. Here we report the emergence of the PANGO lineage C.1.2, detected at low prevalence in South Africa and eleven other countries. The initial C.1.2 detection is associated with a high substitution rate, and includes changes within the spike protein that have been associated with increased transmissibility or reduced neutralization sensitivity in SARS-CoV-2 variants of concern or variants of interest. Like Beta and Delta, C.1.2 shows significantly reduced neutralization sensitivity to plasma from vaccinees and individuals infected with the ancestral D614G virus. In contrast, convalescent donors infected with either Beta or Delta show high plasma neutralization against C.1.2. These functional data suggest that vaccine efficacy against C.1.2 will be equivalent to Beta and Delta, and that prior infection with either Beta or Delta will likely offer protection against C.1.2.

SARS-CoV-2 prolonged infection during advanced HIV disease evolves extensive immune escape.

Characterizing SARS-CoV-2 evolution in specific geographies may help predict properties of the variants that come from these regions. We mapped neutralization of a SARS-CoV-2 strain that evolved over 6 months from ancestral virus in a person with advanced HIV disease in South Africa; this person was infected prior to emergence of the Beta and Delta variants. We longitudinally tracked the evolved virus and tested it against self-plasma and convalescent plasma from ancestral, Beta, and Delta infections. Early virus was similar to ancestral, but it evolved a multitude of mutations found in Omicron and other variants. It showed substantial but incomplete Pfizer BNT162b2 escape, weak neutralization by self-plasma, and despite pre-dating Delta, it also showed extensive escape of Delta infection-elicited neutralization. This example is consistent with the notion that SARS-CoV-2 evolving in individual immune-compromised hosts, including those with advanced HIV disease, may gain immune escape of vaccines and enhanced escape of Delta immunity, and this has implications for vaccine breakthrough and reinfections.

Molecular basis of immune evasion by the Delta and Kappa SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission leads to the emergence of variants, including the B.1.617.2 (Delta) variant of concern that is causing a new wave of infections and has become globally dominant. We show that these variants dampen the in vitro potency of vaccine-elicited serum neutralizing antibodies and provide a structural framework for describing their immune evasion. Mutations in the B.1.617.1 (Kappa) and Delta spike glycoproteins abrogate recognition by several monoclonal antibodies via alteration of key antigenic sites, including remodeling of the Delta amino-terminal domain. The angiotensin-converting enzyme 2 binding affinities of the Kappa and Delta receptor binding domains are comparable to the Wuhan-Hu-1 isolate, whereas B.1.617.2+ (Delta+) exhibits markedly reduced affinity.

Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines.

Understanding vaccine-elicited protection against SARS-CoV-2 variants and other sarbecoviruses is key for guiding public health policies. We show that a clinical stage multivalent SARS-CoV-2 spike receptor-binding domain nanoparticle (RBD-NP) vaccine protects mice from SARS-CoV-2 challenge after a single immunization, indicating a potential dose-sparing strategy. We benchmarked serum neutralizing activity elicited by RBD-NPs in non-human primates against a lead prefusion-stabilized SARS-CoV-2 spike (HexaPro) using a panel of circulating mutants. Polyclonal antibodies elicited by both vaccines are similarly resilient to many RBD residue substitutions tested, although mutations at and surrounding position 484 have negative consequences for neutralization. Mosaic and cocktail nanoparticle immunogens displaying multiple sarbecovirus RBDs elicit broad neutralizing activity in mice and protect mice against SARS-CoV challenge even in the absence of SARS-CoV RBD in the vaccine. This study provides proof of principle that multivalent sarbecovirus RBD-NPs induce heterotypic protection and motivates advancing such broadly protective sarbecovirus vaccines to the clinic.

SARS-CoV-2 immune evasion by the B.1.427/B.1.429 variant of concern.

A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429), which was originally detected in California, carries spike glycoprotein mutations S13I in the signal peptide, W152C in the N-terminal domain (NTD), and L452R in the receptor-binding domain (RBD). Plasma from individuals vaccinated with a Wuhan-1 isolate-based messenger RNA vaccine or from convalescent individuals exhibited neutralizing titers that were reduced 2- to 3.5-fold against the B.1.427/B.1.429 variant relative to wild-type pseudoviruses. The L452R mutation reduced neutralizing activity in 14 of 34 RBD-specific monoclonal antibodies (mAbs). The S13I and W152C mutations resulted in total loss of neutralization for 10 of 10 NTD-specific mAbs because the NTD antigenic supersite was remodeled by a shift of the signal peptide cleavage site and the formation of a new disulfide bond, as revealed by mass spectrometry and structural studies.

High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV-2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity.

SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1-20 µM range in vitro. Furthermore, Exebryl-1, a ß-amyloid anti-aggregation molecule for Alzheimer's therapy, was shown to have antiviral activity between 10 to 66 µM, in Vero 76, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral.

In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19.

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N = 80 samples from persons with RT-PCR confirmed SARS-CoV-2 infection), and a specificity of 97.2% (N = 106 control samples).

Backbone chemical shift assignments for the SARS-CoV-2 non-structural protein Nsp9: intermediate (ms - µs) dynamics in the C-terminal helix at the dimer interface.

The Betacoronavirus SARS-CoV-2 non-structural protein Nsp9 is a 113-residue protein that is essential for viral replication, and consequently, a potential target for the development of therapeutics against COVID19 infections. To capture insights into the dynamics of the protein's backbone in solution and accelerate the identification and mapping of ligand-binding surfaces through chemical shift perturbation studies, the backbone 1H, 13C, and 15N NMR chemical shifts for Nsp9 have been extensively assigned. These assignments were assisted by the preparation of an ~ 70% deuterated sample and residue-specific, 15N-labelled samples (V, L, M, F, and K). A major feature of the assignments was the "missing" amide resonances for N96-L106 in the 1H-15N HSQC spectrum, a region that comprises almost the complete C-terminal α-helix that forms a major part of the homodimer interface in the crystal structure of SARS-CoV-2 Nsp9, suggesting this region either undergoes intermediate motion in the ms to µs timescale and/or is heterogenous. These "missing" amide resonances do not unambiguously appear in the 1H-15N HSQC spectrum of SARS-CoV-2 Nsp9 collected at a concentration of 0.0007 mM. At this concentration, at the detection limit, native mass spectrometry indicates the protein is exclusively in the monomeric state, suggesting the intermediate motion in the C-terminal of Nsp9 may be due to intramolecular dynamics. Perhaps this intermediate ms to µs timescale dynamics is the physical basis for a previously suggested "fluidity" of the C-terminal helix that may be responsible for homophilic (Nsp9-Nsp9) and postulated heterophilic (Nsp9-Unknown) protein-protein interactions.